title: A novel homology model of TRPC3 reveals allosteric coupling between gate and selectivity filter.
abstract: Utilizing a novel molecular model of TRPC3, based on the voltage-gated sodium channel from Arcobacter butzleri (Na(V)AB) as template, we performed structure-guided mutagenesis experiments to identify amino acid residues involved in divalent permeation and gating. Substituted cysteine accessibility screening within the predicted selectivity filter uncovered amino acids 629-631 as the narrowest part of the permeation pathway with an estimated pore diameter of < 5.8Å. E630 was found to govern not only divalent permeability but also sensitivity of the channel to block by ruthenium red. Mutations in a hydrophobic cluster at the cytosolic termini of transmembrane segment 6, corresponding to the S6 bundle crossing structure in Na(V)AB, distorted channel gating. Removal of a large hydrophobic residue (I667A or I667E) generated channels with approximately 60% constitutive activity, suggesting I667 as part of the dynamic structure occluding the permeation path. Destabilization of the gate was associated with reduced Ca2+ permeability, altered cysteine cross-linking in the selectivity filter and promoted channel block by ruthenium red. Collectively, we present a structural model of the TRPC3 permeation pathway and localize the channel's selectivity filter and the occluding gate. Moreover, we provide evidence for allosteric coupling between the gate and the selectivity filter in TRPC3.
authors.affiliation: Institute of Pharmaceutical Sciences--Pharmacology and Toxicology, University of Graz, A-8010 Graz, Austria.
authors.forename: Michaela
authors.initials: M
authors.lastname: Lichtenegger
authors.forename: Thomas
authors.initials: T
authors.lastname: Stockner
authors.forename: Michael
authors.initials: M
authors.lastname: Poteser
authors.forename: Hannes
authors.initials: H
authors.lastname: Schleifer
authors.forename: Dieter
authors.initials: D
authors.lastname: Platzer
authors.forename: Christoph
authors.initials: C
authors.lastname: Romanin
authors.forename: Klaus
authors.initials: K
authors.lastname: Groschner
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D020640:Arcobacter / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D012430:Ruthenium Red / Q000494:pharmacology; D055672:Static Electricity; D050052:TRPC Cation Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D012430:Ruthenium Red; D002118:Calcium
keywords: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 1-stearoyl-2-arachidonoyl-sn-glycerol; BAPTA; DAG; G protein coupled receptor; GPCR; Gating; Homology modeling; PLC; SAG; Selectivity; TRPC3; diacylglycerol; phospholipase C